Changes in transcriptional regulation can affect the level, timing, and spatial expression of genes and consequently, the phenotypic outcome of development. In fact, such changes are associated with a variety of congenital disorders in humans. The proposed project utilizes the sea urchin as a model system to explore the functional consequence of changes in genes and their cis-regulatory sequences with regard to protein-binding affinity, patterns of gene expression in the embryo, and/or phenotype. Thus, Specific Aim 1 is to determine the extent to which there is variation in the cis-regulatory region of SM50 in S. purpuratus (the "purple urchin"), and to evaluate the role of natural selection in producing this variation. This will be accomplished by isolating this DNA sequence from multiple individuals as well as from multiple populations of the same species, S. purpuratus. A variety of statistical tests will then be performed to examine the evolutionary forces that have produced variation in the cis-regulatory region of SM50. Specific Aim 2 is to determine the extent to which there are differences in the cis-regulatory region of SM50 among closely related species of sea urchins, and to evaluate their functional significance. This will be accomplished by isolating the DNA sequence from several species in the Family Strongylocentrotidae, as well as two species belonging to a different family. Experiments will then be performed to assess the impact of differences on embryonic development. For example, GFP reporter constructs will be utilized to confirm the ability of the DNA sequence to activate transcription in cases where the cis-regulatory region of SM50 is significantly different from S. purpuratus. Finally, Specific Aim 3 is to determine the extent to which there have been changes in the cis-regulatory "linkages" of genes responsible for development of the larval skeleton among distantly related species of sea urchins, and to evaluate their functional significance. As a first step, the investigator will isolate skeletogenic genes from E. tribuloides (the "pencil urchin") and examine their expression pattern in the embryo through in situ hybridization. Future work would focus on changes in transcriptional regulation that may have produced differences in gene expression between S. purpuratus and E. tribuloides. It is the investigator's goal to eventually correlate such changes with alterations in the origin and behavior of spicule-forming cells during embryonic development.